The primary objective of Project 1 is to define the mechanisms of functional impairment of HCV-specific CDS T cells associated with viral persistence. While viral escape mutations within T cell epitopes occur in HCV immune evasion, a significant proportion of CDS T cells in chronically infected subjects recognize HCV epitopes that do not demonstrate escape mutations. It is thus likely that functional impairment of CDS cells specific for nonescaped epitopes is an additional important factor in HCV persistence. We propose that a comprehensive comparative analysis of HCV-specific CDS T cell phenot5TDe and function among patients who clear HCV infection versus those who do not, and between T cells recognizing epitopes that undergo substitution and those that do not will reveal specific molecular and cellular mechanisms of T cell unresponsiveness relevant to viral persistence. Specifically, we aim l)To perform a set of in vitro fiinctional analyses assessing the capacity of HCV specific CDS T cells of various previously characterized phenotj^jes to produce relevant effector cj^okines and to perform killer functions and 2)To develop and characterize an in vivo cj^otoxic lymphocyte (CTL) assay for functional analysis of tetramer+ HCV specific CDS cells using adoptive transfer into an established NOD/SCID/--/- system. This will allow us to directly analyze the in vivo functional effects of antibodies and/or cytokines targeted at potentially relevant cell membrane receptors on human HCV specific CDS T cells. Using specific antagonist antibodies, candidate molecular determinants of CDS T cell unresponsiveness will be interrogated in this novel in vivo system in which human T cells from patients are adoptively transferred into receptive immunodeficient mice. Outcomes of this interrogation will have direct translational relevance to the immunotherapy of chronic HCV infection as well as enhancing understanding of T cell impairment associated with persistent infection. Results of studies of humoral immune responses from Project 2 will be integrated to expand knowledge of the interplay between humoral and cellular immune responses to HCV in humans. We have already demonstrated that we can obtain the critical specimens required for this investigation and will be able to conduct unique longitudinal studies of adaptive immune responses in acute HCV.